Systemic Errors in Quantitative Polymerase Chain Reaction Titration of Self-Complementary Adeno-Associated Viral Vectors and Improved Alternative Methods

Abstract

Self-complementary AAV (scAAV) vector genomes contain a covalently closed hairpin derived from a mutated inverted terminal repeat which connects the two monomer single stranded genomes into a head-to-head or tail-to-tail dimer. We found that during quantitative PCR (qPCR) this structure inhibits the amplification of proximal amplicons and causes the systemic underreporting of copy number by as much as 10-fold. We show that cleavage of scAAV vector genomes with restriction endonuclease to liberate amplicons from the covalently closed terminal hairpin restores quantitative amplification, and we implement this procedure in a simple, modified qPCR titration method for scAAV vectors. Additionally, we developed and present an AAV genome titration procedure based on gel electrophoresis that requires minimal sample processing and has very low inter assay variability, and as such is well suited for the rigorous quality control demands of clinical vector production facilities.

Limitations of Encapsidation of Recombinant Self-Complementary Adeno-Associated Viral Genomes in Different Serotype Capsids and Their Quantitation

“…1 and 2). Although the discrepancy was modest with WT serotypes capsid and short viral genomes, which is consistent with the observation by Fagone et al (2012), the measured titer could be as much as fivefold different with capsid-mutant vectors and larger genomes. One of our initial concerns was the different denaturing conditions for vector genomes prior to DNA slot-blot and Southern blot assays.…”

“…During the course of this trial, however, it became apparent that the qPCR method used to determine the vector titers led to an underestimate by a factor of *10 due to the presence of covalently closed hairpins at one end of the scAAV vector DNA and rapid self-annealing of the viral genomes (Fagone et al, 2012). Consequently, the use of primers close to the intact inverted terminal repeats (ITRs) during qPCR assays is recommended, when titering regular scAAV genomes (< 2.4 kb) encapsidated in WT AAV serotype capsids.…”

“…A universal realtime PCR assay involving the AAV ITR sequences for determining the vector titers has been reported (Aurnhammer et al, 2012), but as stated above, one must exercise caution to rely solely on this method in view of the observation made by Kapranov et al (2012) that large amounts of DNA sequences close to the ends of viral genomes are encapsidated in AAV vectors. Recently, gel titration assays based on DNAstaining methodology have also been described to provide robust and reliable quantitation (Fagone et al, 2012). However, the method requires large amounts of purified vectors and thus is more suitable for clinical-grade vector preparations.…”

“…The main drawback of these two assays is that they do not provide information about viral genome integrity. It has been reported that qPCR assays may result in substantial differences in determining the titers of self-complementary AAV (scAAV) vectors, depending on the localization of the primer sets in the scAAV genomes (Fagone et al, 2012). The discrepancy could be nearly 10-fold (Allay et al, 2011).…”

Gene Therapy for Rare Diseases: Summary of a National Institutes of Health Workshop, September 13, 2012

“…Even comparing dosing across trials may be a challenge. For example, in the hemophilia trial that used a self-complementary AAV8 vector, the initial dose was discovered to be almost 10-fold higher than originally measured because the quantitative polymerase chain reaction assay did not accurately detect the copy number due to the secondary structure (self complementary) of the vector genomes (Fangone et al, 2012).…”